The overarching goal of the present proposal is to elucidate the molecular mechanisms by which cellular cofactors control the distribution of murine leukemia virus (MLV) integration sites in chromatin. The selection of chromosomal targets for retroviral integration is not random and varies markedly for different retroviral gener. For example, the gamma-retroviruses including MLV favor integration near transcription start sites and CpG islands, whereas lentiviruses including HIV-1 preferentially integrate within active genes. These observations have suggested that different cellular binding partners of retroviral integrases could be responsible for distinct integration site selectivity. However, until very recently, only one example has been reported: lens epithelium-derived growth factor (LEDGF/p75), which functions as a bimodal tether that engages HIV-1 intasomes and navigates them to active genes. The significance of exploring the molecular mechanisms of gamma-retroviral MLV integration site selectivity is exemplified by the development of MLV-based vectors for human gene-therapy. In clinical trials, the use of gamma-retroviral vectors to correct primary immunodeficiencies has been curative, but adverse events have occurred associated with insertional activation of protooncogenes by MLV-based vectors. We have recently discovered that the bromodomain and extra terminal domain (BET) proteins (Brd2, 3, 4) are the principal cellular binding partners of MLV IN and demonstrated their significance for targeting MLV integration at transcription start sites. The present application aims to extend these important initial findings to better understand the underlying mechanism for how BET proteins selectively recognize MLV IN and navigate the gamma-retroviral integration to specific sites in chromatin. In particular, aim 1 will study structural and mechanistic foundations for how MLV IN recognizes BET proteins selectively and with high affinity; and aim 2 will examine a proposed bimodal mechanism for BET proteins-mediated link between MLV integration and select chromatin sites. Our experiments are expected to yield important novel findings, which will benefit a wide scientific community interested in understanding molecular mechanisms of retroviral integration. Since BET proteins-MLV IN interactions are only the second system, besides HIV-1 IN-LEDGF/p75 interactions, our findings will elucidate important mechanistic similarities and differences between MLV and HIV-1 integration site selectivity. Thus, the proposed studies will lead to better understanding of how different retroviruses have evolved to utilize distinct cellular chromatin binding partners to insert their genetic material at select sits in the host chromosome. Additionally, our findings will lead to better understanding of molecular mechanisms for integrations of MLV-based vectors near proto-oncogenes during human gene-therapy trails, which have been linked to significant adverse outcomes in patients.